1. Field of the Invention
The invention generally relates to plasmepsin inhibitors and the treatment of malaria. In particular, the invention provides methods and compositions for the use of peptidomimetic allophenylnorstatine based inhibitors of the antimalarial target aspartyl protease Plasmepsin II. More generally, the invention provides compounds and methods for inhibiting plasmepsins (e.g. Plasmepsin I, Plasmepsin II, Plasmepsin IV, HAP) which may provide a number of different pharmaceutical and medical benefits.
2. Background of the Invention
Malaria is one of the most serious infectious diseases in the world, affecting close to 300 million individuals each year. It has been estimated that approximately 40% of the world population lives in regions where malaria is endemic. Each year between 1 and 1.5 million people, mainly children, die from malaria, a number that is continuously increasing due to the proliferation of parasites that are resistant to conventional drug therapies (Wyler, 1993). The rapid spread of drug resistant parasites clearly underscores the need for new therapies and consequently the identification of novel targets for drug development. The malaria parasite uses the hemoglobin of the infected victim as a source of nutrients and energy. One of the key enzymes involved in the degradation of hemoglobin is Plasmepsin II, an aspartic protease of 37 kDa. Since the inhibition of this enzyme leads to starvation of the parasite, Plasmepsin II has been acknowledged to be an important target for the development of new antimalarials.
Four species of protozoan parasites of the genus Plasmodium (P. falciparum, P. vivax, P. malariae and P. ovale) are responsible for malaria in humans; P. vivax is the most common but P. falciparum causes the most fatalities (Butler et al., 1997; Miller et al., 1994). The Plasmodium parasite invades red blood cells and consumes tip to 75% of their hemoglobin content (Goldberg, 1993). The process takes place in an acidic digestive vacuole in the parasite. Three enzymes that digest hemoglobin have been identified in the food vacuole, one cysteine protease (falcipain) and two aspartic proteases (Plasmepsin I and Plasmepsin II) (Francis et al., 1997b). The inhibition of any of these enzymes leads to the starvation of the parasite and has been proposed as a viable strategy for drug development Plasmepsin I and Plasmepsin II are 73% sequence identical. They have different substrate specificities and both contribute to the degradation of hemoglobin. Plasmepsin I is synthesized and processed to a mature form soon after the parasite invades the red blood cell, while the appearance of Plasmepsin II occurs later in development (Francis et al., 1997a). The expression and production of active recombinant Plasmepsin I has been shown to be difficult, yielding a truncated protein that lacks the kinetic properties of the native enzyme (Luker et al., 1996). Plasmepsin U, on the other hand, has been successfully expressed, the recombinant protein behaves identically to the protein isolated from the parasite and its high resolution structure has been determined by x-ray crystallography (Luker et al., 1996; Silva et al., 1996). For those reasons, Plasmepsin II is the target of choice for structure-based drug design, even though the targeting of Plasmepsin I and other plasmepsins is also expected.
Plasmepsin II is a protein of 37 kDa (329 amino acids). The crystallographic structure of Plasmepsin II in complex with the generic statine-based aspartic protease inhibitor pepstatin A (IvaValValStaAlaSta) has been obtained at 2.7 Å for the Plasmodium falciparum enzyme (pdb file 1 sme) (Silva et al., 1996) and 2.5 Å for the Plasmodium vivax enzyme (pdb file 1 qs8). Plasmepsin II has the typical bilobal structure and topology of eukaryotic aspartic proteases. The active site is located at the interface between the two lobes and is partially covered by a characteristic β-hairpin structure known as the flap. The secondary structure of Plasmepsin II is predominantly beta with only a small fraction (˜10%) of amino acids in alpha-helix. Even though pepstatin A and other related statine-containing peptides are known to inhibit Plasmepsin II and other aspartic proteases, very few non-peptidic inhibitors have been described. A common problem with these inhibitors is their poor selectivity and discrimination versus the human aspartic protease Cathepsin D. Cathepsin D is a human protease in the endosomal-lysosomal pathway involved in lysosomal biogenesis and protein targeting, it has 35% overall sequence homology and even higher binding site homology with Plasmepsin II, thus representing a target that needs to be avoided in the development of Plasmepsin II inhibitors.
Allophenylnorstatine-based compounds have been described before in relation to the development of HIV-1 protease inhibitors (Kiso, 1996; Kiso, 1998; Kiso et al., 1999; Mimoto et al., 1999). These compounds are characterized by containing a unique unnatural amino acid, allophenylnorstatine ((2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid) containing a hydroxymethylcarbonyl isostere. Some of these compounds have been shown to be high affinity inhibitors of the HIV-1 protease, they have low toxicity and excellent bioavailability (Kiso, 1996; Kiso, 1998; Kiso et al., 1999; Mimoto et al., 1999).